Bioelectronics is a progressing interdisciplinary research field that involves the integration of biomaterials with electronic devices. Bioelectronics methods have been used for analyzing cells and assaying biological molecules and cells. In one type of application, cells are cultured on microelectrodes and cell-electrode impedance or cell-substrate impedance is measured and analyzed to monitor cellular changes, such as changes in cell morphology. For example, PCT Application No. PCT/US03/22557, titled “Impedance Based Devices and Methods for Use in Assays”, discloses a device for detecting cells and/or molecules on an electrode surface. The device detects cells and/or molecules through measurement of impedance changes resulting from the attachment or binding of cells and/or molecules to the electrode surfaces. A number of embodiments of the device are disclosed, together with the apparatuses, and systems for using such devices to perform certain cell based assays.
Bringing a new drug to the market can take anywhere between 8 to 16 years, and the average cost of developing a drug is now around $500-$800 million with the cost expected to hit the $1 billion mark within the next four years. Cardiotoxicity has been cited as the reason for 30 percent of all failed drug compounds during development and is a major cause of compound attrition. The late detection of cardiotoxic side effects caused by pharmacological compounds can impede drug discovery and development projects, and consequently increase their cost. Testing for the potential cardiotoxic side effects of compounds at an early stage of drug development has therefore been the goal of many pharmaceutical and biotechnology companies. Cardiotoxicity itself can entail a number of short-term and long term cellular events including directly affecting the beating rate of cardiomyocytes, viability of cardiomyocytes and morphology of cardiomyocytes as would occur in hypertrophy. The core of the current issue in pharmacological safety assessment and drug development is the lack of a reliable screening methodology capable of monitoring potential drug-mediated cardiotoxicity and distinguishing between different modes of cardiotoxicity. What is urgently needed in the field is a good cell-based model system as well as a monitoring system with a physiological and functional readout that can provide incisive information regarding potential cardiotoxic side effects of drugs.
Traditionally, the drug discovery industry has undertaken two different approaches for toxicological assessment of drug candidate leads in cardiac function. The first approach involves isolation of cardiomyocytes directly from a mammalian species such as rats and dogs followed by electrophysiological and viability studies on the isolated cardiomyocytes. This approach is extremely labor-intensive, time consuming and costly and at the same time not very amenable to the high throughput demands of pharmaceutical industry
An alternative method for prediction of cardiotoxicity of drug candidate leads early in the drug development process has involved utilizing cell-based assay models which heterologously express specific ion channels such as hERG channels or voltage-gated calcium channels. These cardiac ion channels have been envisioned as possible molecular targets through which drugs could induce cardiotoxicity. These cell-based systems allow the assessment of drug-channel interaction by monitoring the effect of the drug on the currents produced by the different channels in cultured cells using a technique known as ‘patch clamping’, which isolates regions of the cell membrane containing channel proteins and measures changes in electrical potential difference. Use of this method in high throughput requires automation of patch clamping in array format, which even though is available in last several years is not yet widespread. Another issue with this approach is that cardiac toxicity may occur by other mechanisms which can easily be missed by this type of targeted approach.
An alternative to the in vitro ion-channel recording assays as well as the labor-intensive isolation of primary tissue is to utilize the differentiation of embryonic stem (ES) cells into cardiomyocytes as a starting material for functional assays. The utility of ES cells as a treatment for various chronic diseases has received much attention in recent years. Mammalian ES cells are self-renewing cells derived from the inner cell mass of a blastocyst stage embryo, which can be differentiated into multiple different cell types. It has been demonstrated that the mouse ES cells as well as human ES cells can be differentiated into cardiomyocytes, which retain the ability to beat in culture.
The differentiation of ES cells first involves an intermediate in vitro developmental stage in which ES cells form compact cell structures known as embryoid bodies. These embryoid bodies can induce the developmental program of ES cell differentiation into multiple cell types including cardiomyocytes, which are distinguished in culture by their ability to undergo spontaneous beating. These ES derived in vitro differentiated cardiomyocytes recapitulates the normal development of cardiomyocytes as evidenced by the stage-specific expression of cardiomyocyte specific genes. All the known transcription factors, ion channels and structural proteins that are part of normal heart development and function in vivo are also expressed in ES-derived cardiomyocyte.
Because ES cells are self-renewing, cells in culture can serve as an excellent source for continuous production of cardiomyocytes. Therefore, these cardiomyocytes which behave in every way like normal cardiomyocytes isolated from the heart tissue itself addresses the ever important supply problem and for the first time allows for assessment of cardiac function and its modulation by lead candidate drugs and compounds in relatively large scale in both viability assays, assessment of morphology and in monitoring the beating function of cardiomyocytes. Furthermore, because the technology exists to selectively knockout or express trans-genes in ES cells, it provides an excellent model system to study the role of certain genes in cardiac development and function without having to be concerned about adverse affects on overall embryonic development in transgenic animals.
The ability to express transgenes in ES cells has been utilized as a way to enrich for preparation of cardiomyocytes that are 100% pure. For example, the gene encoding GFP has been cloned downstream of a cardiac-specific promoter and then introduced into ES cells. Embryoid cells, which ultimately differentiate into cardiomyocytes, will therefore express the GFP transgenes and these cells can be easily isolated by cell sorting techniques and therefore an enriched cardiomyocyte population can be obtained.
Technologies designed to assess cardiomyocyte behavior and function and the effect of drugs and other manipulations in vitro can be divided into two different approaches. One approach involves long term assessment of cardiomyocyte viability for example in response to certain compounds. Such assays are typically end point assays designed to measure a cellular component such as ATP, which correlates with the degree of viability of the cells. The other approach involves studying short term effect of drugs and compounds on beating function of cardiomyocytes. High throughput techniques for short term functional characterization of ion channels and other targets in cardiomyocytes has been rather challenging and limited. Systems such as automatic patch clamp instrumentation that are available can monitor a single cardiomyocyte at a time and with very limited throughput.
In US 2011/0039294 an approach to monitoring a cardiomyocyte population is disclosed, which includes both impedance monitoring and extracellular recording technologies with high precision. While acceptance of the technology is increasing, there still remain challenges when working with the cells themselves. For example, when using primary cardiomyocyte cells harvested from tissue or after extended culturing, the beating of cells can stop or slow. Accordingly, there is a need to provide devices, systems and methods that assess the cardiotoxicity of compounds while working to ensure the continued or regular beating of cardiomyocyte cells.